PROJECT SUMMARY Defects in DNA damage response and DNA repair are the driving forces of genomic instability and tumorigenesis. Gaining a better understanding of the pathways involved in DNA repair not only increases our understanding of cancer etiology, but also provides new targets for cancer therapies. A key protein involved in DNA repair and tumorigenesis is p53-binding protein 1, i.e. 53BP1. My laboratory has been working on 53BP1 for many years. Our group was one of the first to demonstrate the role of 53BP1 in DNA damage response. We established the first 53bp1 knockout (KO) mice; we also revealed that 53BP1 is required for DNA repair and acts as a tumor suppressor in vivo. In addition, we elucidated the regulation of 53BP1 after DNA damage. We and others demonstrated that the H2AX-dependent DNA damage signaling pathway controls the recruitment and accumulation of 53BP1 at sites of DNA breaks. However, 53BP1 can also localize to DNA damage sites in an H2AX-independent manner, although the underlying mechanisms remain to be determined. Moreover, we showed that 53BP1 is critical for a particular repair process called class-switch recombination, indicating that 53BP1 is involved in a special DNA repair pathway that is distinctly different from the canonical nonhomologous end-joining pathway. Our recent studies and those of others suggest that 53BP1 suppresses homologous recombination repair in BRCA1-deficient cells, which is critically important for response to poly (ADP-ribose) polymerase inhibitor-based cancer therapies. Together, these data highlight the importance of 53BP1 in counteracting homologous recombination repair in response to DNA damage. In this proposal, we plan to focus on 53BP1 and elucidate at the molecular level how 53BP1 is regulated after DNA damage and contributes to DNA repair and genome maintenance. To further understand the regulation of 53BP1 localization and function at DNA damage sites, we recently performed tandem affinity purification coupled with mass spectrometry analysis to identify proteins that would specifically associate with a region of 53BP1, which is necessary and sufficient for its localization to DNA damage sites. Surprisingly, we uncovered several novel 53BP1-binding proteins, including NUDT16, NUDT16L1, and DEK. In this proposal, we will 1) further determine the roles of NUDT16L1 and NUDT16 in 53BP1 regulation and in the DNA damage response, and 2) elucidate the functional significance of DEK and other newly discovered 53BP1-associated proteins in damage-induced 53BP1 localization, DNA repair, and the maintenance of genomic integrity. These studies will help us understand the key components that act upstream of 53BP1 and function together with 53BP1 in DNA repair and genome maintenance.